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Effluents from urban wastewater treatment plants (WWTP) consist of complex mixtures of substances that can affect processes in the receiving ecosystems. Some of these substances (toxic contaminants) stress biological activity at all concentrations, while others (e.g., nutrients) subsidize it at low concentrations and stress it above a threshold, causing subsidy-stress responses. Thus, the overall effects of WWTP effluents depend mostly on their composition and the dilution capacity of the receiving water bodies. We assessed the immediate and legacy effects of WWTP effluents in artificial streams, where we measured the uptake of soluble reactive phosphorus (SRP) by the biofilm, biomass accrual, benthic metabolism and organic matter decomposition (OMD). In a first phase (32 d), the channels were subjected to a gradient of effluent contribution, from pure stream water to pure effluent. WWTP effluent affected the ecosystem processes we measured, although we found no clear subsidy-stress patterns except for biofilm biomass accrual. Instead, most of the processes were subsidized, although they showed complex and process-specific patterns. Benthic metabolism and OMD were subsidized without saturation, as they peaked at medium and high levels of pollution, respectively, but they never fell below control levels. SRP uptake was the only process that decreased with increasing effluent concentration. In a second phase of the experiment (23 d), all channels were kept on pure stream water to analyse the legacy effects of the effluent. For most of the processes, there were clear legacy effects, which followed either subsidy, stress, or subsidy-stress patterns. SRP uptake capacity was stressed with increasing pollution legacy, whereas algal accrual and benthic metabolism continued being subsidized. Conversely, biofilm biomass accrual and OMD showed no legacy effects. Overall, the WWTP effluent caused complex and process-specific responses in our experiment, mainly driven by the mixed contribution of subsidizers and stressors. These results help improving our understanding of the effects of urban pollution on stream ecosystem functioning. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved. This research was supported by the European Union 7th Framework Programme (GLOBAQUA; 603629-ENV-2013-6.2.1). Authors also acknowledge the financial support from the University of the Basque Country (pre-doctoral fellowship to O. Pereda), the Basque Government (Consolidated Research Group: Stream Ecology 7-CA-18/10), and the Economy and Knowledge Department of the Catalan Government (Consolidated Research Group: ICRA-ENV 2017 SGR 1124). Authors are also especially grateful to Maria Casellas, Carme Font, Carmen Gutiérrez, Ferran Romero and Laia Sabater-Liesa for their assistance during the laboratory experiments.

Spatial heterogeneity along river networks is interrupted by dams, affecting the transport, processing, and storage of organic matter, as well as the distribution of biota. We here investigated the structure of planktonic (free-living, FL), particle-attached (PA) and sediment-associated (SD) bacterial and archaeal communities within a small reservoir. We combined targeted-amplicon sequencing of bacterial and archaeal 16S rRNA genes in the DNA and RNA community fractions from FL, PA and SD, followed by imputed functional metagenomics, in order to unveil differences in their potential metabolic capabilities within the reservoir (tail, mid, and dam sections) and lifestyles (FL, PA, SD). Both bacterial and archaeal communities were structured according to their life-style preferences rather than to their location in the reservoir. Bacterial communities were richer and more diverse when attached to particles or inhabiting the sediment, while Archaea showed an opposing trend. Differences between PA and FL bacterial communities were consistent at functional level, the PA community showing higher potential capacity to degrade complex carbohydrates, aromatic compounds, and proteinaceous materials. Our results stressed that particle-attached prokaryotes were phylogenetically and metabolically distinct from their free-living counterparts, and that performed as hotspots for organic matter processing within the small reservoir Tis study was funded by the project of the Spanish Ministry of Economy and Competitiveness SPACESTREAM (CGL2017-88640-C2-1-R). The authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124), and to R. Marcé, D.von Schiller, C. Gutierrez, J. P. Casas-Ruiz, L. Gómez-Gener, V. Acuña and B. Obrador for their help during the sampling. ICRA is part of the CERCA program

Freshwater ecosystems are exposed to multiple stressors, but their individual and combined effects remain largely unexplored. Here, we investigated the response of stream biofilm bacterial communities to warming, hydrological stress, and pesticide exposure. We used 24 artificial streams on which epilithic (growing on coarse sediments) and epipsammic (growing on fine sediments) stream biofilms were maintained. Bacterial community composition and estimated function of biofilms exposed during 30 days to individual and combined stressors were assessed using 16S rRNA gene metabarcoding. Among the individual effects by stressors, hydrological stress (i.e., a simulated low-flow situation) was the most relevant, since it significantly altered 57% of the most abundant bacterial taxa (n = 28), followed by warming (21%) and pesticide exposure (11%). Regarding the combined effects, 16% of all stressor combinations resulted in significant interactions on bacterial community composition and estimated function. Antagonistic responses prevailed (57 to 89% of all significant interactions), followed by synergisms (11 to 43%), on specific bacterial taxa, indicating that multiple-stressor scenarios could lead to unexpected shifts in the community composition and associated functions of riverine bacterial communities. IMPORTANCE Freshwater ecosystems such as rivers are of crucial importance for human well-being. However, human activities result in many stressors (e.g., toxic chemicals, increased water temperatures, and hydrological alterations) cooccurring in rivers and streams worldwide. Among the many organisms inhabiting rivers and streams, bacteria are ecologically crucial; they are placed at the base of virtually all food webs and they recycle the organic matter needed for bigger organisms. Most of these bacteria are in close contact with river substratum, where they form the biofilms. There is an urgent need to evaluate the effects of these stressors on river biofilms, so we can anticipate future environmental problems. In this study, we experimentally exposed river biofilms to a pesticide mixture, an increase in water temperature and a simulated low-flow condition, in order to evaluate the individual and joint effects of these stressors on the bacterial community composition and estimated function.

Temporary streams are characterized by the alternation of dry and wet hydrological phases, creating both a harsh environment for the biota as well as a high diversity of opportunities for adaptation. These systems are mainly microbial-based during several of these hydrological phases, and those growing on all solid substrata (biofilms) accordingly change their physical structure and community composition. Biofilms experience large decreases in cell densities and biomass, both of bacteria and algae, during dryness. Algal and bacterial communities show remarkable decreases in their diversity, at least locally (at the habitat scale). Biofilms also respond with significant physiological plasticity to each of the hydrological changes. The decreasing humidity of the substrata through the drying process, and the changing quantity and quality of organic matter and nutrients available in the stream during that process, causes unequal responses on the biofilm bacteria and algae. Biofilm algae are affected faster than bacteria by the hydric stress, and as a result the ecosystem respiration resists longer than gross primary production to the increasing duration of flow intermittency. This response implies enhancing ecosystem heterotrophy, a pattern that can be exacerbated in temporary streams suffering of longer dry periods under global change This work has received a grant from the European Community 7th Framework Programme undergrant agreement No. 603629-ENV-2013-6.2.1-Globaqua.The authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government as being part of the Consolidated Research Group of the Catalan Institute for Water Research (2014SGR291)

The brown food chain (based on decomposers) co-exists in streams with the green food chain (based on primary producers). The two trophic chains perform specific ecosystem functions which may be altered by the effect of contaminants. Copper is a common contaminant with recognized effects on several compartments of the two trophic chains. We applied it in two separate mesocosm experiments, in which we tested the effects of copper after contrasting patterns of contaminant exposure (constant vs hump-shaped). The constant input simulated a chronic contamination (average of 20 μg/L Cu), while the hump-shaped simulated the steady arrival of copper, the occurrence of a peak (reaching ca. 60 μg/L Cu), and its progressive decrease (down to 10–15 μg/L Cu). In the green trophic food chain, copper exposure decreased the total chlorophyll-a as well as the basal fluorescence and the photosynthetic yield. The treatment receiving hump-shaped inputs caused the highest mortality of the green food chain consumer, the snail Radix balthica. In the chronic copper exposure, mortality achieved a maximum of 80% by the end of the experiment but occurred later than that in the hump-shaped treatment. Effects on the brown food chain were not so pronounced; the microbial decomposition rate of leaflitter decreased nearly ca. 50% after 14 days of copper exposure. Effects on decomposition translated into the ingestion performance of detritivores, which decreased in the two copper treatments. Our results provide evidence that copper affected the two trophic food chains. The hump-shaped arrival included a peak of high concentration, which caused lethal effects on the consumers, but also a decreasing limb, which allowed a partial recovery of the algal photosynthetic variables. Our results suggest the need to consider the different compartments and functions performed within the stream trophic web when evaluating the effects of a contaminant in a river ecosystem.

Human appropriation of water resources may induce water stress in freshwater ecosystems when ecosystem needs are not met. Intensive abstraction and regulation cause river ecosystems to shift towards non-natural flow regimes, which might have implications for their water quality, biological structure and functioning. We performed a meta-analysis of published studies to assess the potential effects of water stress on nutrients, microcontaminants, biological communities (bacteria, algae, invertebrates and fish), and ecosystem functions (organic matter breakdown, gross primary production and respiration). Despite the different nature of the flow regime changes, our meta-analysis showed significant effects of human-driven water stress, such as significant increases in algal biomass and metabolism and reduced invertebrate richness, abundance and density and organic matter decomposition. Water stress also significantly decreased phosphate concentration and increased the concentration of pharmaceutical compounds. The magnitude of significant effects was dependent on climate, rainfall regime, period of the year, river size and type of water stress. Among the different causes of water stress, flow regulation by dams produced the strongest effects, followed by water abstraction and channelization. © 2018, The Author(s)., This project was funded by the European Commission under the grant No. 603629 – project GLOBAQUA. VF acknowledges financial support from the Portuguese Foundation for Science and Technology (FCT) through UID/MAR/04292/2013 and IF/00129/2014. The authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through the Consolidated Research Group (ICRA-ENV 2017 SGR 1124).

An increasing amount of carbon-based nanomaterials (CNM) (mostly fullerenes, carbon nanotubes and graphene) has been observed in aquatic systems over the last years. However, the potential toxicity of these CNM on aquatic ecosystems remains unclear. This paper reviews the existing literature on the toxic effects of CNM in aquatic organisms as well as the toxic effects of CNM through influencing the toxicity of other micro-pollutants, and outlines a series of research needs to reduce the uncertainty associated with CNMs toxic effects. The results show that environmental concentrations of CNM do not pose a threat on aquatic organisms on their own. The observed concentrations of CNM in aquatic environments are in the order of ngL-1 or even lower, much below than the lowest observed effect concentrations (LOEC) on different aquatic organisms (in the order of mgL-1). Toxic effects have been mainly observed in short-term experiments at high concentrations, and toxicity principally depends on the type of organisms, exposition time and CNM preparation methods. Moreover, we observed that CNM interact (establishing synergistic and/or antagonistic effects) with other micro-pollutants. Apparently, the resulting interaction is highly dependent on the chemical properties of each micro-pollutant, CNM acting either as carriers or as sorbents, thereby modifying the original toxicity of the contaminants. Results stress the need of studying the interactive effects of CNM with other micro-pollutants at environmental relevant concentrations, as well as their effects on biological communities in the long-term.

The potential for rivers to alter the flux of dissolved organic matter (DOM) from land to ocean is widely accepted. Yet anticipating when and where rivers behave as active reactors vs. passive pipes of DOM stands as a major knowledge gap in river biogeochemistry, resulting in uncertainties for global carbon models. Here, we investigate the controls on in-stream DOM dynamics by evaluating changes in DOM concentration and composition along several reaches of a medium-sized river network over one full hydrological year. Roughly half of the observations over time and space showed active reactor conditions and, among these, similar proportion of gains and losses was measured. High water residence times promoted the active over passive behavior of the reaches, while DOM properties and nitrate availability determined whether they supplied or removed DOM from the river. Among different DOM fractions, protein-like DOM both of terrestrial and aquatic origin seemed to drive bulk DOM patterns. Our study emphasizes the role of water residence time as a physical constraint for in-stream processes, and provides new insights into the key factors governing the net balance between in-stream gains and losses of DOM in rivers.

Fullerenes are carbon nanomaterials that have awaken a strong interest due to their adsorption properties and potential applications in many fields. However, there are some gaps of information about their effects and bioconcentration potential in the aquatic biota. In the present work, freshwater biofilms and snails (Radix sp.) were exposed to fullerene C60 aggregates, at concentrations in the low μg/L order, in mesocosms specifically designed to mimic the conditions of a natural stream. The bioconcentration factors of C60 fullerene and its main transformation product, [6,6]C60O epoxide, were studied to the mentioned organisms employing analyses by liquid chromatography coupled to high-resolution mass spectrometry. Our results show that C60 fullerene and its [6,6]C60O present a low bioconcentration factor (BCF) to biofilms: BCFC60 = 1.34 ± 0.95 L/kgdw and BCFC60O = 1.43 ± 0.72 L/kgdw. This suggests that the sorption of these aggregates to biota may be less favoured than it would be suggested by its hydrophobic character. According to our model, the surface of fullerene aggregates is saturated with [6,6]C60O molecules, which exposes the polar epoxide moieties in the surface of the aggregates and decreases their affinity to biofilms. In contrast, freshwater snails showed a moderate capacity to actively retain C60 fullerenes in their organism (BAFC60 = 2670 ± 3070 L/kgdw; BAFC60O = 1330 ± 1680 L/kgdw), probably through ingestion. Our results indicate that the bioaccumulation of these carbon nanomaterials can be hardly estimated using their respective octanol-water partition coefficients, and that their colloidal properties, as well as the feeding strategies of the tested organism, play fundamental roles. © 2019 Elsevier Inc., This work was supported by the Spanish Ministry of Economy and Competitiveness through the project NANO-transfer (ERA-NET SIINN PCIN-2015-182-CO2-01) and by the Generalitat de Catalunya (Consolidated Research Groups “2014 SGR 418 – Water and Soil Quality Unit” and “2014 SGR 291 – ICRA”). Lúcia H.M.L.M. Santos acknowledges the Juan de la Cierva program (IJCI-2017-32747) and Julio C. López-Doval thanks the program Juan de la Cierva from the Spanish Ministry of Economy and Competitiveness (IJCI-2015-23644). Appendix A

The current knowledge on bioaccumulation of emerging contaminants (ECs) in aquatic invertebrates exposed to the realistic environmental concentrations is limited. Even less is known about the effects of chemical pollution exposure on the metabolome of aquatic invertebrates. We conducted an in situ translocation experiment with passive filter-feeding caddisfly larvae (Hydropsyche sp.) in an effluent-influenced river in order to i) unravel the bioaccumulation (and recovery) dynamics of ECs in aquatic invertebrates, and ii) test whether exposure to environmentally realistic concentrations of ECs will translate into metabolic profile changes in the insects. The experiment was carried out at two sites, upstream and downstream of the discharge of an urban wastewater treatment plant effluent. The translocated animals were collected at 2-week intervals for 46 days. Both pharmaceuticals and endocrine disrupting compounds (EDCs) were detected in water (62 and 7 compounds, respectively), whereas in Hydropsyche tissues 5 EDCs accumulated. Overall, specimens from the upstream site translocated to the impacted site reached higher ECs concentrations in their tissues, as a reflection of the contaminants’ water concentrations. However, bioaccumulation was a temporary process susceptible to change under lower contaminant concentrations. Non-targeted metabolite profiling detected fine metabolic changes in translocated Hydropsyche larvae. Both translocations equally induced stress, but it was higher in animals translocated to the impacted site.

Emerging pollutants occur in complex mixtures in rivers and have the potential to interact with freshwater organisms. The chronic effects of nominal exposure to 3 μg/L of fullerenes (C60) and 1 μg/L of triclosan (TCS) alone and in a binary mixture, were evaluated using the freshwater snail Radix balthica. Pollutants accumulation, reproductive output and feeding behavior were selected as sublethal endpoints. After 21 days of exposure, we did not observe interactive effects between TCS and C60 on the studied endpoints, except for the accumulation of C60 in R. balthica in TCS + C60 treatment, which was lower than when the fullerenes were alone. Neither TCS nor C60 caused significant effects on reproduction, expressed as number of eggs per individual, but an increase in the clutch size was observed in treatments with TCS at the third week of exposure, independently of the presence of C60 (16.15 ± 1.67 and 18.9 ± 4.01 eggs/egg mass in TCS and TCS + C60 treatments, respectively, vs. 13.17 ± 4.01 in control). The presence of C60 significantly enhanced the grazing activity of R. balthica during the first seven days (4.95 ± 1.35 and 3.91 ± 0.59% of the area grazed per individual in C60 and TCS + C60 treatments, respectively, vs 2.6 ± 0.39% in control). The accumulation of TCS was quite similar in treatments where this pollutant was present (BAF ≈ 1007 L/kg d.w.); however, the accumulation of C60 was higher when the nanoparticles were alone (BAF = 254.88 L/kg d.w.) than when it was in the binary mixture (BAF = 7.79 L/kg d.w). Overall, although TCS has been listed as an endocrine disrupter compound, no significant effects on reproduction were observed in the assayed conditions. Regarding C60, the limited effects on feeding activity and the low BAF obtained in this experiment indicate that fullerenes do not have ecological consequences of relevance at the studied environmental concentrations in freshwater snails. © 2019 Elsevier Inc., This work was supported by the Spanish Ministry of Economy and Competitiveness through the project NANO-transfer (ERA-NET SIINN PCIN-2015-182-CO2-01) and by the Generalitat de Catalunya (Consolidated Research Groups “2014 SGR 418 – Water and Soil Quality Unit” and “2014 SGR 291 – ICRA”). Julio C. López-Doval (IJCI-2015-23644), Lúcia H.M.L.M. Santos (FJCI-2014-22377 and IJCI-2017-32747) and Anna Freixa (FJCI-2017-33171) acknowledge the Juan de la Cierva program from the Spanish Ministry of Economy and Competitiveness. Sara Rodriguez-Mozaz acknowledges the Ramon y Cajal program (RYC-2014-16707).

A huge variety of organic microcontaminants are presently detected in freshwater ecosystems, but there is still a lack of knowledge about their interactions, either with living organisms or with other contaminants. Actually, carbon nanomaterials like fullerenes (C60) can act as carriers of organic microcontaminants, but their relevance in processes like bioaccumulation and biotransformation of organic microcontaminants by organisms is unknown. In this study, mesocosm experiments were used to assess the bioaccumulation and biotransformation of three organic microcontaminants (venlafaxine, diuron and triclosan) in river biofilms, and to understand how much the concomitant presence of C60 at environmental relevant concentrations could impact these processes. Results indicated that venlafaxine exhibited the highest bioaccumulation (13% of the initial concentration of venlafaxine in water), while biotransformation was more evident for triclosan (5% of the initial concentration of triclosan in water). Furthermore, biotransformation products such as methyl-triclosan were also present in the biofilm, with levels up to 42% of the concentration of accumulated triclosan. The presence of C60 did not involve relevant changes in the bioaccumulation and biotransformation of microcontaminants in biofilms, which showed similar patterns. Nevertheless, the study shows that a detailed evaluation of the partition of the organic microcontaminants and their transformation products in freshwater systems are important to better understand the impact of the co-existence of others microcontaminants, like carbon nanomaterials, in their possible routes of bioaccumulation and biotransformation. © 2018 Elsevier Inc., This work has been funded by the Spanish Ministry of Economy and Competitiveness (project NANOTRANSFER; ERA SIINN PCIN-2015-182-C02-02 and project PLAS-MED; CTM2017-89701-C3-2-R). Authors also acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124 & 2017 SGR 1404). Lúcia H.M.L.M. Santos acknowledges the Juan de la Cierva Program (FJCI-2014-22377) and Sara Rodríguez-Mozaz acknowledges the Ramon y Cajal Program (RYC-2014-16707). Funding sources This work has been funded by the Spanish Ministry of Economy and Competitiveness (project NANOTRANSFER; ERA SIINN PCIN-2015-182-C02-02 and project PLAS-MED; CTM2017-89701-C3-2-R). Authors also acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124 & 2017 SGR 1404). Lúcia H.M.L.M. Santos acknowledges the Juan de la Cierva program (FJCI-2014-22377) and Sara Rodríguez-Mozaz acknowledges the Ramon y Cajal program (RYC-2014-16707).

Pharmaceuticals are designed to improve human and animal health, but may also be a threat to freshwater ecosystems, particularly after receiving urban or wastewater treatment plant (WWTP) effluents. Knowledge on the fate and attenuation of pharmaceuticals in engineered and natural ecosystems is rather fragmented, and comparable methods are needed to facilitate the comprehension of those processes amongst systems. In this study the dynamics of 8 pharmaceuticals (acetaminophen, sulfapyridine, sulfamethoxazole, carbamazepine, venlafaxine, ibuprofen, diclofenac, diazepam) and 11 of their transformation products were investigated in a WWTP and the associated receiving river ecosystem. During 3 days, concentrations of these compounds were quantified at the influents, effluents, and wastage of the WWTP, and at different distances downstream the effluent at the river. Attenuation (net balance between removal and release from and to the water column) was estimated in both engineered and natural systems using a comparable model-based approach by considering different uncertainty sources (e.g. chemical analysis, sampling, and flow measurements). Results showed that pharmaceuticals load reduction was higher in the WWTP, but attenuation efficiencies (as half-life times) were higher in the river. In particular, the load of only 5 out of the 19 pharmaceuticals was reduced by more than 90% at the WWTP, while the rest were only partially or non-attenuated (or released) and discharged into the receiving river. At the river, only the load of ibuprofen was reduced by more than 50% (out of the 6 parent compounds present in the river), while partial and non-attenuation (or release) was observed for some of their transformation products. Linkages in the routing of some pharmaceuticals (venlafaxine, carbamazepine, ibuprofen and diclofenac) and their corresponding transformation products were also identified at both WWTP and river. Finally, the followed procedure showed that dynamic attenuation in the coupled WWTP-river system could be successfully predicted with simple first order attenuation kinetics for most modeled compounds This research was supported by the European Communities 7th Framework Programme Marie Curie Career Integration Grant PCIG9-GA-2011-293535. Authors also acknowledge the support from the Economy and Knowledge Department of the Catalan Government (Consolidated Research Group 2014 SGR 291 - ICRA) and the Spanish Ministry of Economy and Competitiveness for funding (CTM2015- 66892-R, CTM2012-38314-C02-01 and RYC-2013-14595)

Most fluvial networks worldwide include watercourses that recurrently cease to flow and run dry. The spatial and temporal extent of the dry phase of these temporary watercourses is increasing as a result of global change. Yet, current estimates of carbon emissions from fluvial networks do not consider temporary watercourses when they are dry. We characterized the magnitude and variability of carbon emissions from dry watercourses by measuring the carbon dioxide (CO2) flux from 10 dry streambeds of a fluvial network during the dry period and comparing it to the CO2 flux from the same streambeds during the flowing period and to the CO2 flux from their adjacent upland soils. We also looked for potential drivers regulating the CO2 emissions by examining the main physical and chemical properties of dry streambed sediments and adjacent upland soils. The CO2 efflux from dry streambeds (mean ± SD = 781.4 ± 390.2 mmol m−2 day−1) doubled the CO2 efflux from flowing streambeds (305.6 ± 206.1 mmol m−2 day−1) and was comparable to the CO2 efflux from upland soils (896.1 ± 263.2 mmol m−2 day−1). However, dry streambed sediments and upland soils were physicochemically distinct and differed in the variables regulating their CO2 efflux. Overall, our results indicate that dry streambeds constitute a unique and biogeochemically active habitat that can emit significant amounts of CO2 to the atmosphere. Thus, omitting CO2 emissions from temporary streams when they are dry may overlook the role of a key component of the carbon balance of fluvial networks This research was funded by the Spanish Ministry of Economy and Competitiveness through the Projects CGL2011-30474-C02-01 and CGL2014-58760-C3-1-R.

We used the freshwater insect Hydropsyche sp. to investigate the impact of diets lacking arachidonic acid (ARA) and an environmentally relevant mixture of NSAIDs (Ibuprofen, Ketoprofen, Diclofenac and Naproxen at a nominal concentration of all compounds together 16.75 μg L) on their metabolism of ARA and prostaglandins (PGs). The organisms were exposed for 16 days to four different treatments: a reference (FF), a diet lacking ARA (O), to NSAIDs in water (FFN) and to the combination of the two factors (ON). Mortality, biomass and bioconcentration of pharmaceuticals were investigated. The ARA and PGs levels in the organisms were monitored by utilising a targeted metabolomics approach. NSAIDs or dietary constraints did not produce significant differences in biomass or mortality of Hydropsyche sp. among treatments. In organisms exposed to NSAIDs, all pharmaceuticals were detected, except for Ketoprofen. Metabolomic approach determined the presence of PGH, PGE and PGD. Levels of ARA diminished significantly in those organisms in treatment ON. The levels of PGs responded negatively to the absence of ARA in diet: PGH diminished significantly with respect to the reference in treatment O while PGE diminished significantly in treatment ON. Regarding the effects of NSAIDs on ARA metabolism, our results suggest that it was sensitive to NSAIDs, but effects were weak and did not imply a general decrease in the PGs. We confirmed that ARA was the main substrate for the synthesis of PGs in Hydropsyche sp, their absence or poor levels of ARA in diet, produced changes in the PG levels., This work was supported by GLOBAQUA project (FP7, No 603629), the Spanish Ministry of Economy and Competitiveness through the projects CLIMALERT (PCIN-2017- 068) and SPACESTREAM (CGL 2017-88640-C2-1-R) and by the Generalitat de Catalunya (Consolidated Research Group “2014 SGR 291 – ICRA”). ICRA researchers thank funding from CERCA program. Julio C. López-Doval thanks the program Juan de la Cierva from the Spanish Ministry of Economy and Competitiveness (IJCI-2015-23644). Albert Serra-Compte benefits from an FI-DGR research fellowship from the Catalan Government (2016FI_B00601). Sara Rodriguez-Mozaz acknowledges the Ramon y Cajal program (RYC- 2014–16707) from the Spanish State Research Agency of Spanish Ministry of Science, Innovation and Universities (AEI-MCIU). Carmen Gutierrez Provecho and Juan David González-Trujillo (ICRA) are gratefully acknowledged for their assistance in the field collections and lab processing of the samples.

Water scarcity is a serious environmental problem in many European regions, and will likely increase in the near future as a consequence of increased abstraction and climate change. Water scarcity exacerbates the effects of multiple stressors, and thus results in decreased water quality. It impacts river ecosystems, threatens the services they provide, and it will force managers and policy-makers to change their current practices. The EU-FP7 project GLOBAQUA aims at identifying the prevalence, interaction and linkages between stressors, and to assess their effects on the chemical and ecological status of freshwater ecosystems in order to improve water management practice and policies. GLOBAQUA assembles a multidisciplinary team of 21 European plus 2 non-European scientific institutions, as well as water authorities and river basin managers. The project includes experts in hydrology, chemistry, biology, geomorphology, modelling, socio-economics, governance science, knowledge brokerage, and policy advocacy. GLOBAQUA studies six river basins (Ebro, Adige, Sava, Evrotas, Anglian and Souss Massa) affected by water scarcity, and aims to answer the following questions: how does water scarcity interact with other existing stressors in the study river basins? How will these interactions change according to the different scenarios of future global change? Which will be the foreseeable consequences for river ecosystems? How will these in turn affect the services the ecosystems provide? How should management and policies be adapted to minimise the ecological, economic and societal consequences? These questions will be approached by combining data-mining, field- and laboratory-based research, and modelling. Here, we outline the general structure of the project and the activities to be conducted within the fourteen work-packages of GLOBAQUA., This work has been supported by the European Communities 7th Framework Programme Funding under Grant agreement no. 603629- ENV-2013-6.2.1-Globaqua and by the Generalitat de Catalunya (Consolidated Research Groups “2014 SGR 418 - Water and Soil Quality Unit” and 2014 SGR 291 - ICRA). Damià Barceló acknowledges support from the Visiting Professor Program of King Saud University. Special thanks are due to all partners of the GLOBAQUA consortium and the peer review panel for ensuring quality results and fruitful collaboration within the project.

The effluents of wastewater treatment plants (WWTPs) include a complex mixture of nutrients and pollutants. Nutrients can subsidise autotrophic and heterotrophic organisms, while toxic pollutants can act as stressors, depending, for instance, on their concentration and interactions in the environment. Hence, it is difficult to predict the overall effect of WWTP effluents on river ecosystem functioning. We assessed the effects of WWTP effluents on river biofilms and ecosystem metabolism in one river segment upstream from a WWTP and three segments downstream from the WWTP and following a pollution gradient. The photosynthetic capacity and enzymatic activity of biofilms showed no change, with the exception of leucine aminopeptidase, which followed the pollution gradient most likely driven by changes in organic matter availability. The effluent produced mixed effects on ecosystem-scale metabolism. It promoted respiration (subsidy effect), probably as a consequence of enhanced availability of organic matter. On the other hand, and despite enhanced nutrient concentrations, photosynthesis-irradiance relationships showed that the effluent partly decoupled primary production from light availability, thus suggesting a stress effect. Overall, WWTP effluents can alter the balance between autotrophic and heterotrophic processes and produce spatial discontinuities in ecosystem functioning along rivers as a consequence of the mixed contribution of stressors and subsidisers This research was supported by the Spanish Ministry of Economy and Competitiveness and FEDER foundings through the SCARCE Consolider-Ingenio CSD2009-00065 and ABSTRACT CGL2012-35848 projects, and the European Communities 7th Framework Programme Funding under Grant agreement no. 603629-ENV-2013-6.2.1-Globaqua. We especially thank the people who assisted in the field and in the laboratory. A thorough review of the manuscript by Prof. Roger I. Jones and two unknown reviewers is deeply appreciated too. We also want to acknowledge financial support in terms of predoctoral grants from the University of the Basque Country (I. Aristi), the Basque Government (M. Arroita), as well as a postdoctoral grant ‘Juan de la Cierva’ (jci-2010-06397) (D. von Schiller) and a Marie Curie European Reintegration Grant (PERG07-GA-2010-259219) (V. Acuña). This work was partly supported by the Basque Government (Consolidated Research Group: Stream Ecology 7-CA-18/10)

Organic micro-contaminants (OMCs) enter in freshwaters and interact with other contaminants such as carbon nanoparticles, becoming a problem of unknown consequences for river ecosystems. Carbon nanoparticles (as fullerenes C60) are good adsorbents of organic contaminants and their interaction can potentially affect their toxicity to river biofilms. We tested the C60 interactions with selected OMCs and their effects on river biofilms in different short-term experiments. In these, river biofilms were exposed to C60 and three OMCs (triclosan, diuron, or venlafaxine) and their respective mixtures with fullerenes (C60 + each OMC). The effects were evaluated on structural, molecular, and functional descriptors of river biofilms. Our results showed that C60 did not cause toxic effects in river biofilms, whereas diuron and triclosan significantly affected the heterotrophic and phototrophic components of biofilms and venlafaxine affected only the phototrophic component. The joint exposure of C60 with venlafaxine was not producing differences with respect to the former response of the toxicant, but the overall response was antagonistic (i.e., decreased toxicity) with diuron, and synergistic (i.e., increased toxicity) with triclosan. We suggest that differences in the toxic responses could be related to the respective molecular structure of each OMC, to the concentration proportion between OMC and C60, and to the possible competition between C60 pollutants on blocking the receptors of the biological cell membranes. We conclude that the presence of C60 at low concentrations modified the toxicity of OMC to river biofilms. These interactions should therefore be considered when predicting toxicity of OMC in river ecosystems. © 2018 Freixa, Acuña, Gutierrez, Sanchís, Santos, Rodriguez-Mozaz, Farré, Barceló and Sabater., We would like to thank Ferran Romero for his help in the molecular analysis, Lorenzo Proia for his help in using MicroResp technique, and Maria Casellas for laboratory assistance.

Aquatic organisms from freshwater ecosystems impacted by waste water treatment plant (WWTP) effluents are constantly exposed to constant concentrations of pharmaceuticals, endocrine disruptors and related compounds, among other anthropogenic contaminants. Macroinvertebrates inhabiting freshwater ecosystems might be useful bioindicators of exposure to contaminants, since their lives are long enough to bioaccumulate, but at the same time may integrate short-term changes in the environment. However, studies about potential bioaccumulation of emerging contaminants in these organisms are very scarce. The objectives of this study were to develop an analytical methodology for the analysis of 41 pharmaceuticals and 21 endocrine disruptors in freshwater invertebrates. In addition, bioaccumulation of these contaminants in three macroinvertebrate taxa inhabiting a waste water treatment plant -impacted river was evaluated. The method for the simultaneous extraction of both families of compounds is based on sonication, purification via removal of phospholipids, and analysis by ultra performance liquid chromatography coupled to a mass spectrometer (UPLC-MS/MS) in tandem. Recoveries for pharmaceuticals were 34-125%, and for endocrine disruptors were 48-117%. Method detection limits (MDLs) for EDCs were in the range of 0.080-2.4 ng g-1, and for pharmaceuticals, 0.060-4.3 ng g-1. These pollutants were detected in water samples taken downstream the waste water treatment plant effluent at concentrations up to 572 ng L-1. Two non-esteroidal anti-inflammatory drugs, diclofenac and ibuprofen, and four endocrine disruptors - estrone, bisphenol A, TBEP, and nonylphenol - were detected in at least one macroinvertebrate taxa in concentrations up to 183 ng g-1 (dry weight). An isobaric interference was identified during the analysis of diclofenac in Hydropsyche samples, which was successfully discriminated via accurate mass determination by TFC-LTQ Orbitrap This study has been financed by Spanish Ministry o fEconomy and Competitiveness through the project SCARCE (Consolider-Ingenio 2010CSD200900065). This work was partly supported by the Generalitat de Catalunya (Consolidated Research Group: Catalan Institute for water Research 2014 SGR 291)

In many arid and semi-arid systems, biological communities in river ecosystems are submitted to flow interruption and desiccation, as well as to the impact of urban wastewaters. In this work, we studied (using a LC-LTQ-Orbitrap) the metabolomic response of biofilm communities exposed to both hydrological and chemical stressors. Fluvial biofilms were exposed to a mixture of 9 pharmaceuticals at a total concentration of 5000ng/L (mimicking concentrations and compounds found in polluted aquatic environments) and/or to seven days of desiccation, under laboratory conditions. The biosynthesis of fatty acids was the main metabolic pathway disrupted in biofilms. Endogenous biofilm's metabolites (metabolome) altered due to these stressors were identified. The metabolites that significantly changed only due to one of the stressors could be proposed as potential specific biomarkers. A biomarker of pharmaceutical exposure was the lysophosphatidic acid, which decreased a 160%, while for desiccation stearidonic acid (increased 160%), 16-Oxohexadecanoic acid (increased 340%) and palmitoleic acid (decreased 290%) were the biomarkers proposed. Besides, other metabolites showed different responses depending on the treatment, such as palmitic acid, linolenic acid, behenic acid, lignoceric acid and azelaic acid. The Carbon:Phosphorus (C:P) molar ratio increased due to all stress factors, whereas the algal community composition changed mainly due to desiccation. A possible relationship between those changes observed in structural parameters and the metabolome of biofilms was explored. Overall, our findings support the use of metabolomics to unravel at molecular level the effects from chemical and physical stressors on complex microbial communities, such as biofilms, and pinpoint biomarkers of exposure.

Biofilms are a key component in the nutrient removal from the water column. However, nutrient uptake by biofilms may be hampered by the occurrence of pollutants or other stressors. This study aimed: (i) to investigate the biofilm phosphorus (P) uptake capacity as a relevant process for the maintenance of fluvial water quality and (ii) to explore the sensitivity of this process to different chemical and environmental stressors. We conducted chamber experiments to test for the relevance of biofilm P uptake capacity (PUC) as a tool to detect effects of pollutants on river self-depuration. PUC was calculated by measuring P temporal decay after performing controlled P-spikes in chambers with biofilm-colonized tiles. Four different experiments were conducted to evaluate the response of PUC to: (a) several river waters from increasing polluted sites; (b) the effect of the bactericide triclosan (TCS); (c) the combined effect of TCS and grazers; and (d) the effect of TCS after a drought episode that affected the biofilms. These experiments showed that biofilms decreased their PUC along the pollution gradient. The biofilm PUC was significantly reduced after receiving high TCS concentrations, though lower TCS concentrations also affected the biofilm when this was submitted to grazing pressure. PUC decrease was induced by flow interruption which further enhanced the TCS negative effects. Overall, PUC was sensitive to the effects of pollutants like TCS as well as to the action of biological (grazing) and environmental (drought) factors. The study also showed that multiple stressors enhance the negative effects of pollutants on the PUC of biofilms. Our study values the use of biofilms’ PUC as a sensitive ecological-based tool to assess the effects of chemicals on freshwater communities and their derived functioning in river ecosystems.

Intermittent rivers and ephemeral streams (IRES) support highly biodiverse primary producers, including algae, cyanobacteria, and aquatic and riparian plants. All these groups share common traits to cope with a harsh environment whose water table varies and where desiccation is common. Traits include morphological characteristics, life cycle adaptations, and physiological mechanisms to contend with hydric stress and increased water temperatures. Despite these environmental challenges, primary producers play ubiquitous roles in the fluxes of energy and material in IRES. They are the basis for ecosystem metabolism, expressed as episodic pulses of production that occur immediately following rewetting, when their abundant output of high-quality organic matter is critical for supporting consumers both in-stream and in the neighboring terrestrial system.

River and stream biofilms in mediterranean fluvial ecosystems face both extreme seasonality as well as arrhythmic fluctuations. The hydrological extremes (droughts and floods) impose direct changes in water availability but also in the quantity and quality of organic matter and nutrients that sustain the microbial growth. This review analyzes how these ecological pulses might determine unique properties of biofilms developing in mediterranean streams. The paper brings together data from heterotrophic and autotrophic community structure, and extracellular enzyme activities in biofilms in mediterranean streams. Mediterranean stream biofilms show higher use of peptides during the favorable period for epilithic algae development (spring), and preferential use of cellulose and hemicellulose in autumn as a response to allochthonous input. The drying process causes the reduction in bacterial production and chlorophyll biomass, but the rapid recovery of both autotrophs and heterotrophs with rewetting indicates their adaptability to fluctuations. Bacteria surviving the drought are mainly associated with sediment and leaf litter which serve as "humid refuges". Some algae and cyanobacteria show resistant strategies to cope with the drought stress. The resistance to these fluctuations is strongly linked to the streambed characteristics (e.g., sediment grain size, organic matter accumulation, nutrient content) This study was funded by the projects CGL2011-30151-C02-01, and SCARCE (Consolider-Ingenio 2010 CSD2009-00065) of the Spanish Ministry of Economy and Competitiveness; the Italy-Spain Exchange Project CNR-CSIC 2006IT0010; and the EU project MIRAGE (FP7-ENV-2007-1 n.211732)

Nutrients and light are the most determinant factors for microbial benthic assemblages in oligotrophic forested streams. We investigated the importance of nutrients and light availability on the structure and the function of epilithic biofilms in a Mediterranean forested stream (Fuirosos, Spain). Biofilms grew on artificial substrata in both enriched and unenriched reaches where shade conditions were simulated. Four different treatments were generated: higher light unenriched, lower light unenriched, higher light enriched (HL-E) and lower light enriched. Chlorophyll a, bacterial density, extracellular polymeric substances (EPS), extracellular leucine aminopeptidase (LAmP) and alkaline phosphatase (APase) activities were analysed during the colonisation at days 4, 9, 16, 22 and 52. At day 52, confocal laser scanning microscopy (CLSM) was used to determine differences in biofilm architecture. CLSM evidenced differences in thickness and structural complexity of biofilms grown in different conditions. Biofilms in HL-E were the thickest and had the most complex structure. The CLSM highlighted that the EPS was agglomerated in the upper layer of enriched-grown biofilms, but evenly distributed through the biofilm in unenriched biofilms. CLSM 3D images suggested that cyanobacteria increased under higher nutrient conditions. Nutrient enrichment caused the decrease of APase activity. Interaction between the two factors affected LAmP activity. HL-E had the highest LAmP and the lowest APase activities, an indication that biofilm responses to nutrients mostly occurred with high-light availability. Our results revealed that the conjoint availability of light and nutrients caused the highest changes in biofilm spatial organisation, microbial structure and functioning in oligotrophic forested streams.

Summary 1. The effects of long-term nutrient addition at moderate levels were examined in the food web of a forested Mediterranean stream. Basal concentrations of N and P were increased twofold (to c. 750 μg N) and threefold (to c. 30 μg P) from ambient concentrations in an experimental reach. Variations in the abundance of microbes (bacteria and algae), meiofauna and macrofauna, microbial processing of organic matter (extracellular enzyme activities) and stoichiometry of biofilms and invertebrates were compared to an upstream control reach during 4 years of artificial nutrient enhancement. 2. Effects were faster in the bacterial compartment but more substantial in the algal compartment. Epilithic algal biomass doubled in the enriched section jointly triggered by nutrients and increased light irradiance in winter and early spring. Only a few animal groups reacted to the enrichment, including the meiofaunal Copepoda, linked to their high use of enriched FPOM, and macrofaunal grazers (Ancylus), which followed the large algal biomass increase. 3. The enrichment caused biofilm phosphatase activity to decrease, while activities related to the use of algal-related materials (peptidase, β-glucosidase) increased. Enzymatic activities related to the use of allochthonous organic matter showed only minor and episodic increases. 4. Changes in stoichiometric ratios were apparent in the epilithic compartment, but not in the sand sediment or in the FPOM. Increases in P content were delayed for 9 months in epilithic biofilms and for nearly 2 years in the case of N. 5. After 2 years of enrichment, the flatworm Schmidtea polychroa (predator), oligochaetes (detritivore) and tadpoles of Bufo bufo (grazer) showed higher per cent N. 6. Enrichment effects were produced in spite of flow cessations that occurred commonly in summer. The results show that forested streams subjected to sustained (though minor) nutrient enrichment changed aspects of their biological structure and metabolism and that changes were especially favoured by periods when light was not limiting.

Mediterranean streams are characterized by water flow changes caused by floods and droughts. When intermittency occurs in river ecosystems, hydrologic connectivity is interrupted and this affects benthic, hyporheic and flowing water compartments. Organic matter use and transport can be particularly affected during the transition from wet to dry and dry to wet conditions. In order to characterize the changes in benthic organic matter quantity and quality throughout a drying and rewetting process, organic matter, and enzyme activities were analyzed in the benthic accumulated material (biofilms growing on rocks and cobbles, leaves, and sand) and in flowing water (dissolved and particulate fractions). The total polysaccharide, amino acid, and lipid content in the benthic organic matter were on average higher in the drying period than in the rewetting period. However, during the drying period, peptide availability decreased, as indicated by decreases in leucine aminopeptidase activity, as well as amino acid content in the water and benthic material, except leaves; while polysaccharides were actively used, as indicated by an increase in β-glucosidase activity in the benthic substrata and an increase in polysaccharide content of the particulate water fraction and in leaf material. During this process, microbial heterotrophs were constrained to use the organic matter source of the lowest quality (polysaccharides, providing only C), since peptides (providing N and C) were no longer available. During the flow recovery phase, the microbial community rapidly recovered, suggesting the use of refuges and/or adaptation to desiccation during the previous drought period. The scouring during rewetting was responsible for the mobilization of the streambed and loss of benthic material, and the increase in high quality organic matter in transport (at that moment, polysaccharides and amino acids accounted for 30% of the total DOC). The dynamics of progressive and gradual drought effects, as well as the fast recovery after rewetting, might be affected by the interaction of the individual dynamics of each benthic substratum: sand sediments and leaves providing refuge for microorganisms and organic matter storage, while on cobbles, an active bacterial community is developed in the rewetting. Since global climate change may favor a higher intensity and frequency of droughts in streams, understanding the effects of these disturbances on the materials and biota could contribute to reliable resource management. The maintenance of benthic substrata heterogeneity within the stream may be important for stream recovery after droughts.

Herbicides affect the structure and functional parameters of fluvial biofilm. Diuron is toxic to primary producers and disrupts endocrine activity. Here, we studied the interaction between this toxicant and several biological compartments in a simple food chain composed of herbivores (the snail Physella [Costatella] acuta) and biofilm. We used indoor experimental channels to which Diuron was added at a realistic concentration (2 mug/L). Bacterial survival and chlorophyll-a and photosynthetic activity were analyzed in the biofilm. We monitored biomass, mortality, reproduction, and motility as end points in the freshwater snail P. acuta. Our results showed that bacterial survival and photosynthetic activity were sensitive to Diuron. Snails were not affected by the herbicide at the concentration tested. No significant interactions between the toxicant and grazers were observed on the biofilm. Reproductive traits, however, were slightly affected, indicating a possible endocrine disruption.

A system of recirculating channels was used in this study to examine the long-term effects (29 d) of environmentally realistic concentrations of the herbicide diuron (from 0.07 to 7 μg L-1) on biofilm communities. The autotrophic activity of biofilms was affected by this herbicide, as reflected by a marked decrease in the photosynthetic efficiency. Diuron exposure also increased chlorophyll-a content and reduced the biovolume of diatom taxa at low concentrations. The effects on bacteria were also remarkable. Bacterial abundance was reduced after a week of exposure to the herbicide at a range of concentrations. Effects were on the number of live bacteria and on the increase in the leucine-aminopeptidase activity. It is suggested that inputs of herbicides to the river ecosystem at low concentrations may cause a chain of effects in the biofilm, which include inhibitory effects on algae but also indirect effects on the relationships between biofilm components. © 2009 Elsevier Ltd. All rights reserved This study was supported by the European Commission project Modelkey (Project 511237-2 GOCE). Additional funds were provided by the European Project Keybioeffects (MRTN-CT-2006-035695) and by the Spanish Ministry of Science and Technology (Project GCL2006-12785/HID) and the VIECO program (009/RN08/011) of the Spanish Ministry of the Environment and Rural and Marine Affairs

We have studied the differences in the organic matter processing and biofilm composition and structure between autoheterotrophic and heterotrophic biofilm communities. Microbial communities grown on artificial biofilms were monitored, following incubation under light and dark conditions and with or without the addition of glucose as a labile organic compound. Glucose addition greatly affected the microbial biofilm composition as shown by differences in 16S rRNA gene fingerprints. A significant increase in β-glucosidase and peptidase enzyme activities were also observed in glucose-amended biofilms incubated in the dark, suggesting an active bacterial community. Light enhanced the algal and bacterial growth, as well as higher extracellular enzyme activity, thereby indicating a tight algal–bacterial coupling in biofilms incubated under illumination. In these biofilms, organic compounds excreted by photosynthetic microorganisms were readily available for bacterial heterotrophs. This algal–bacterial relationship weakened in glucose-amended biofilms grown in the light, probably because heterotrophic bacteria preferentially use external labile compounds. These results suggest that the availability of labile organic matter in the flowing water and the presence of light may alter the biofilm composition and function, therefore affecting the processing capacity of organic matter in the stream ecosystem.

In Mediterranean forested streams, the large input of particulate organic matter (POM) in autumn structures the benthic microbial community into two interconnected habitats: that associated with the POM itself (leaves and branches) and that colonizing gravel and sandy substrata. Relationships were examined between microbial decomposition activities (β-glucosidase, β-xylosidase, cellobiohydrolase, phenoloxidase, peroxidase, peptidase and phosphatase) at different stream substrata (leaves, branches, sand and gravel) and total carbon (C), nitrogen (N) and phosphorus (P) content of attached microbial communities and stream water matter fractions (particulate and dissolved nutrient concentrations). Microbial communities associated to leaves and branches showed higher C:N and lower N:P molar ratios (averaging 21.7 ± 1.4 and 5.4 ± 2.1, respectively) and higher polysaccharide degrading activity (sum of β-glucosidase, β-xylosidase and cellobiohydrolase activities). Instead, biofi lms on sand and gravel, where algae accumulate and fi ne particulate material were more available, showed lower C:N and higher N:P molar ratios (averaging 10.3 ± 0.6 and 19.9 ± 3.7, respectively) and greater ligninolytic (sum of phenol oxidase and peroxidase activities) and peptidase activities. These results suggest that enzyme activities of microbial attached communities are linked to their nutrient molar ratios and, at the same time, these might be modulated by the different nature of available organic matter (OM) in each substratum and the microbial groups accumulated (algae, bacteria, fungi, microand meiofauna). However, similarities (C:N) and divergences (N:P) between stream water and nutrient molar ratios in microbial communities may also affect nutrient demands and in consequence, the expression of extracellular enzymes. Our results show a relationship between function (extracellular enzyme activities) and nutrient molar ratios of attached microbial communities.

Extracellular polymeric substances (EPS) contribute to biofilm stability and adhesion properties. The EPS matrix might also be a site for free extracellular enzyme activity; however, little is known about participation of enzyme activity in EPS during biofilm formation. In this study, we analyzed the activities of beta-glucosidase, leu-aminopeptidase, and beta-glucosaminidase during the colonization of artificial substrata (glass tiles) in a stream distinguishing enzyme activity in EPS matrix (matrix-enzymes) and total biofilm extracellular enzyme activity. The 1-h incubation of a biofilm suspension and cation-exchange resin followed by centrifugation seems appropriate to extract the matrix fraction (supernatant) and measure matrix enzymes (including free and linked to EPS) in freshwater biofilms, although there is a methodological limitation for using a biofilm suspension instead of an undisrupted biofilm. Total biofilm activities and matrix-enzyme activities showed similar capabilities to decompose organic matter compounds, with a greater capacity for peptide decomposition (leu-aminopeptidase) than for polysaccharides (beta-glucosidase), and a low decomposition of chitin and peptidoglycan (beta-glucosaminidase). Matrix-enzyme activity increased with colonization time, but more slowly than that of total enzyme activity. At the beginning of the colonization experiment (days 1-4) matrix enzymes accounted for 65-81% of total biofilm enzyme activity. Higher proportion of polysaccharides in EPS versus total biofilm, and higher matrix-enzyme activities per microgram of polysaccharides in the EPS were measured during the first 1-3 days of biofilm formation, indicating a high rate of enzyme release into the matrix during this period. Relative contribution of matrix-enzyme activities decreased as biofilm matures, but was maintained at 13-37% of total enzyme activity at the 42- to 49-day-old biofilm. These enzymes, retained and conserved in the EPS, may contribute to community metabolism. When analyzing extracellular enzymes in biofilms, the contribution of matrix enzymes must be considered, especially for young biofilms.

We studied biochemical changes in biofilm and suspended particulate and dissolved organic matter (OM) during the leaf emergence period (March–May 2008) in a forested headwater stream in response to a long-term (4 years, 2004–2008) experimental nutrient enrichment study. This study compared results from one reach upstream of the enrichment point and one reach downstream using moderate nutrient concentrations (nitrogen, N, from 388 to 765 μg L−1 and phosphorus, P, from 10 to 30 μg L−1, resulting in N:P ratios of 85–56). During the spring of 2008, we analysed the chlorophyll content, elemental composition (carbon, C, and N), bacterial density, and extracellular enzyme activities along with their biochemical composition (amino acids, fatty acids and sterols) on biofilm and OM. Nutrients caused changes in the biochemical composition of the biofilm, while changes in the OM were subtle. The C:N ratio of the biofilm decreased with nutrient enrichment likely due to the increase in protein (non-essential amino acids). The polysaccharide and total and essential fatty acid contents were higher when nutrient enrichment coincided with greater light availability. The peptidase extracellular activity was higher in the fertilised reach at early spring, while phosphatase activity decreased at late spring. The suspended and dissolved OM composition did not change due to the nutrient addition, likely due to the lower water residence time in the reach. Headwater systems are highly dynamic, and the biochemical composition of the biofilm changed in response to changes in nutrients but also to light in this study. These changes, although moderate, could influence higher trophic levels through modifications in their diet. This experiment exemplifies how small land use shifts may affect headwater streams This work has been funded by the Spanish Ministry of Economy and Competitiveness with projects CGL2014-58760-C3-R

Interactions between emerging contaminants and other drivers of community structure and function are poorly known. We used laboratory microcosms to investigate the single and combined effects of grazing and triclosan toxicity on the structure and function of stream periphyton. Grazing alone strongly reduced algal biomass, but also reduced oxidative stress and increased periphyton productivity and phosphorus (P) uptake capacity. This suggests that grazed algal communities can compensate for reduced biomass by enhanced growth after being released from grazing. Triclosan exposure (11.6 ± 1.1 μg L−1) reduced the detoxification capacity and P-uptake capacity of periphyton and altered diatom taxonomic composition. This indicates that triclosan at environmentally relevant concentrations affects the capacity of periphyton to remove dissolved nutrients and to cope with toxicant mixtures commonly occurring in streams. Triclosan exposure and grazing pressure had negative synergistic effects on algal size-class distribution and diatom mortality, since the effects of triclosan were higher than expected when periphyton was subject to grazing. Periphyton exposed to toxic substances such as triclosan had a lower capacity to cope with grazing than unexposed communities, because toxicity can limit algal regrowth after release from grazing and promote the loss of less abundant species. This synergism may have important implications because grazing pressure will magnify the negative effects of toxicants on community structure and ecosystem functions such as primary production and nutrient cycling This study was supported by the European Commission through the project Modelkey (Project 511237-2 GOCE). Additional funds were provided by the European Keybioeffects Project (MRTN-CT-2006-035695), VIECO (009/RN08/011), SCARCE (Consolider-Ingenio 2010, CSD2009- 00065) and Fluvialmultistress (CTM2009-14111-CO2-01) and MPCUdG2016/120

Land use type, physical and chemical stressors, and organic microcontaminants were investigated for their effects on the biological communities (biofilms and invertebrates) in several Mediterranean rivers. The diversity of invertebrates, and the scores of the first principal component of a PCA performed with the diatom communities were the best descriptors of the distribution patterns of the biological communities against the river stressors. These two metrics decreased according to the progressive site impairment (associated to higher area of agricultural and urban-industrial, high water conductivity, higher dissolved organic carbon and dissolved inorganic nitrogen concentrations, and higher concentration of organic microcontaminants, particularly pharmaceutical and industrial compounds). The variance partition analyses (RDAs) attributed the major share (10%) of the biological communities' response to the environmental stressors (nutrients, altered discharge, dissolved organic matter), followed by the land use occupation (6%) and of the organic microcontaminants (2%). However, the variance shared by the three groups of descriptors was very high (41%), indicating that their simultaneous occurrence determined most of the variation in the biological communities This study has been financially supported by the EU through the FP7 project GLOBAQUA (Grant agreement No 603629). The authors are part of the Consolidated Research Groups of the Generalitat de Catalunya (2014 SGR 291dICRA and 2014 SGR 418Water and Soil Quality Unit, IDAEA-CSIC)

Organic matter inputs, transport, and storage, ecosystem metabolism, and organic C turnover length were measured in a forested Mediterranean 3rd-order stream (Fuirosos) over a period of 3 y. Meteorological patterns influenced the organic matter dynamics of Fuirosos through 2 pathways: summer weather and flood pulses. Summer rains affected the timing of litter fall and the extent of flow intermittency, which gave rise to accumulations of organic matter in the streambed in dry years. With the onset of flow, these organic matter accumulations were the energy sources for considerable ecosystem respiration (30 g O2 m−2 d−1). Interflood periods punctuated by flood pulses determined cycles of steady accumulation and abrupt removal of stored organic matter. During the interflood periods, the efficiency of organic matter processing increased continuously. The seasonal changes in the riparian forest influenced the ecological consequences of the flood pulses. Resilience of the ecosystem, measured in terms of...

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After indications from the literature that nutrient concentrations may modify the toxicity of herbicides to natural periphyton communities, this study aims to provide experimental proof for atrazine. In this microcosm experiment, phosphate (P) addition did not ameliorate atrazine toxicity to periphyton. Three weeks of P addition did not increase atrazine tolerance (measured as EC50 in acute toxicity tests), whereas exposure to atrazine under conditions that were either P-limited or non-P-limited clearly reduced the development of algal biomass. Long-term exposure to atrazine induced tolerance of the community to the herbicide, and this was not influenced by P addition. Tolerance induction in this microcosm experiment has been compared with previously published field data from the same area of study and indicates that tolerance induction by atrazine may take place under atrazine exposure in streams as well as in microcosms.

The effects of the intensity of seasonal droughts on stream ecosystems were studied in the Fuirosos, an intermittent forested Mediterranean stream. Macroinvertebrate community structure and stream ecosystem metabolism were measured during seasonal summer droughts in 2001, 2002, and 2003. Ecosystem metabolism was profoundly affected by stream intermittency. Organic matter that accumulated during the dry period enhanced ecosystem respiration during the postdrought recovery. Highest biotic diversity was found at low water levels as the stream dried and contracted. Macroinvertebrate community response to drying was stepped and apparently defined by thresholds of transition from drying to cessation of flow and from the dry phase to restoration of flow. Environmental conditions changed markedly with cessation of flow, causing large changes in community structure during 2001 and 2003 (dry years). Drying caused an increase in macroinvertebrate density that peaked in isolated pools soon after flow ceased, ...

The structural characteristics and microprofiles of dissolved oxygen and redox potential were examined in benthic cyanobacterial mats, which developed in a nutrient-rich river. The mats were highly dynamic both in their structure and relative composition, and showed large spatial heterogeneity. The mats differed in the contribution of Oscillatoria limosa, which was higher in the free-floating than in the attached mats. Maximum dissolved oxygen concentration at saturating irradiances occurred within the first 2 mm in the attached mat, but was smaller and deeper in the free-floating mat. The free-floating mats showed heterogeneous profiles inside the same mat due to varying thickness in nearby zones of the mats. In these mats, some black micropatches (high proportion of Oscillatoria) showed anoxic conditions under low irradiances, whereas other brown micropatches (lower proportion of Oscillatoria) never became anoxic. After several hours in the dark, a negative redox potential (− 400 mV) was detect...

Two different streams receiving high nutrient content were compared in relation to evidence provided by structural (biomass) and functional (community metabolism) descriptors. The first (Gaià) was an unshaded agricultural stream, where high net community metabolism and chlorophyll concentration were only modified by flood episodes which caused the sloughing of the benthic community. The second stream (Fuirosos) was forested and nutrients were added for 44 days. In this case, bacteria and macroinvertebrates were also observed and were compared to a control upstream stretch with low nutrient content. Even though there was an increase of chlorophyll because of the nutrient addition, the macroinvertebrate community density as a whole was not affected. Moreover, metabolism (net community metabolism and respiration) did not experience significant changes. An unexpected effect of nutrient addition was detected using a finer scale of observation: during a colonization experiment, chlorophyll and bacterial density became uniform for the different substrata analyzed (sand, cobbles) after 44 days. These structural changes implied a loss of structural heterogeneity which may be associated with significant modifications of stream functioning (such as the nutrient retention) for these systems in the longer term.

Nutrients were artificially increased in an oligotrophic, forested stream by adding nitrogen (as nitrate and ammonia) and phosphorus (as reactive phosphate) continuously for 44 days. This addition increased the nutrient concentrations by 10 (phosphorus), 6 (ammonia) and 4 times (nitrates) with respect to basal concentrations. The effect of the enrichment on the structure and metabolism in benthic habitats (rocks, sand and leaf litter) was studied during a period of moderate light availability (maximum light at the streambed ranged between 20-120 μE m -2 s -1 ), prior to leaf occurrence in the riparian vegetation. A BACI design was used to determine the significance of the addition by comparing an upstream non-enriched (control) reach with a downstream enriched one (impacted). Nutrient addition caused a 3-fold increase in chlorophyll density on rocks and sand, and a remarkable increase in algal cell density, but did not cause changes in the community composition of the algal community. The increase in algal biomass was not reflected in higher net community metabolism (NCM) when this was measured as oxygen production, but higher carbon incorporation was detected at the end of the experiment. Nutrient addition did not significantly alter bacterial cell density on rocks, sand and leaf litter substrata. Accordingly, no substantial increase in community respiration (CR) was detected. Neither did heterotrophic activity (extracellular enzymes) change significantly because of addition, except for a decrease (although weak) in phosphatase in sand. Enrichment did not cause a general increase in macroinvertebrate density or biomass, but some taxa increased in the enriched reach. Scrapers (Ancylus fluviatilis) and detritivores (Oligochaeta, Lumbriculidae significantly augmented their density or biomass on certain substrata. Overall, enhanced nutrient availability was able to cause a remarkable biomass increase in the autotrophic component of the stream, in spite of the moderate light availability.

Aquatic microfungi play a fundamental role in organic matter decomposition in fluvial ecosystems. These micro-organisms degrade leaf recalcitrant compounds like lignin, thereby enhancing the utilization of organic material by the microbial community. The main input of allochthonous organic matter in Mediterranean streams occurs during the autumn. In-stream breakdown processes can be affected by high physical abrasion during flooding but changes in stream water chemistry may also affect decomposition enzymatic activities of stream microorganisms. We measured two ligninolytic activities (phenol oxidase and peroxidase) and a cellulolytic activity (cellobiohydrolase) in leaves, branches, sand and gravel substrata in a reach of a Mediterranean stream. Highest ligninolytic activities were measured in biofilm developed on inorganic substrata (sand and gravel) where also accumulated the highest fungal biomass (ranging from 3.3 x 10 -4 to 7.22 mg Ergosterol gAFDM -1 ) especially in sand substrata. Conversely, cellulolytic activities were significantly higher in biofilm on organic substrata (leaves and branches). Physical and chemical factors, such as discharge and stream water nutrient concentration (Dissolved Inorganic Nitrogen) were affecting enzymatic activities, particularly enhancing the phenol oxidase. Moreover, the chemical composition of the available OM (high cellulose in leaves, high lignin in detritic material) strongly influenced the decomposition activity in each biofilm. A precise description and quantification of the benthic substrata was used to obtain enzymatic activity values in terms of stream reach. Those results showed a temporal pattern in the decomposition activities in the reach, beginning with the decomposition of cellulose (October) followed by lignin compounds (November and December).

We investigated the effect of benthic substratum type (sand and rocks) and nutrient supply (N and P) on biofilm structure and heterotrophic metabolism in a field experiment in a forested Mediterranean stream (Fuirosos). Rock and sand colonization and biofilm formation was intensively studied for 44 d at two stream reaches: control and experimental (continuous addition of phosphate, ammonia, and nitrate). Structural (C, N, and polysaccharide content and bacterial and chlorophyll density) and metabolic biofilm parameters (b-glucosidase, peptidase, and phosphatase enzyme activities) were analyzed throughout the colonization process. The epilithic biofilm (grown on rocks) had a higher peptidase activity at the impacted reach, together with a higher algal and bacterial biomass. The positive relationship between the peptidase activity per cell and the N content of the epilithic biofilm suggested that heterotrophic utilization of proteinaceous compounds from within the biofilm was occurring. In contrast, nutrient addition caused the epipsammic biofilm (grown on sand) to exhibit lowerb-glucosidase and phosphatase activities, without a significant increase in bacterial and algal biomass. The differential response to nutrient addition was related to different structural characteristics within each biofilm. The epipsammic biofilm had a constant and high C : N ratio (22.7) throughout the colonization. The epilithic biofilm had a higher C : N ratio at the beginning of the colonization (43.2) and evolved toward a more complex structure (high polysaccharide content and low C : N ratio) during later stages. The epipsammic biofilm was a site for the accumulation and degradation of organic matter: polysaccharides and organic phosphorus compounds had higher degradation activities.

Summary 1. Single-station diel oxygen curves were used to monitor the oxygen metabolism of an intermittent, forested third-order stream (Fuirosos) in the Mediterranean area, over a period of 22 months. Ecosystem respiration (ER) and gross primary production (GPP) were estimated and related to organic matter inputs and photosynthetically active radiation (PAR) in order to understand the effect of the riparian forest on stream metabolism. 2. Annual ER was 1690 g O2 m−2 year−1 and annual GPP was 275 g O2 m−2 year−1. Fuirosos was therefore a heterotrophic stream, with P : R ratios averaging 0.16. 3. GPP rates were relatively low, ranging from 0.05 to 1.9 g O2 m−2 day−1. The maximum values of GPP occurred during a few weeks in spring, and ended when the riparian canopy was fully closed. The phenology of the riparian vegetation was an important determinant of light availability, and consequently, of GPP. 4. On a daily scale, light and temperature were the most important factors governing the shape of photosynthesis–irradiance (P–I) curves. Several patterns could be generalised in the P–I relationships. Hysteresis-type curves were characteristic of late autumn and winter. Light saturation responses (that occurred at irradiances higher than 90 μE m−2 s−1) were characteristic of early spring. Linear responses occurred during late spring, summer and early autumn when there was no evidence of light saturation. 5. Rates of ER were high when compared with analogous streams, ranging from 0.4 to 32 g O2 m−2 day−1. ER was highest in autumn 2001, when organic matter accumulations on the streambed were extremely high. By contrast, the higher discharge in autumn 2002 prevented these accumulations and caused lower ER. The Mediterranean climate, and in its effect the hydrological regime, were mainly responsible for the temporal variation in benthic organic matter, and consequently of ER.

Toxicity endpoints (nonspecific cytotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, and mutagenicity) were studied in cyanobacterial mats obtained from a shallow river. Some of the cyanobacterial mats tested were known to be non-geosmin producers, while others were geosmin-producers. No microcystin-like compounds were detected by HPLC in any of the biofilm samples. The mutagenicity and neurotoxicity of biofilm metabolites was negligible, and generally weak adverse effects of biofilm extracts detected in a battery of in-vitro assays indicated relatively low human health risks associated with biofilm toxicity. While the toxicity responses detected in the studied biofilms were weak, effects were not related to production of geosmin. It was therefore concluded that the production of this metabolite cannot be taken as an indication per se of the existence of a health hazard.

The ecological factors that co-occur with the production of geosmin have been studied in a Mediterranean river, which shows repeated episodes of this metabolite causing nuisance off flavours. The geosmin peaks consistently occurred from early winter (late January or February) to late spring (May or June). Absolute geosmin maximum were different between years, the highest concentration occurring on March 1999 (146 ng.l -1 ). These episodes coincided with the progressive development of thick algal mats on sediments and cobbles, especially in pools and littoral zones. The dominant taxa in these mats were Oscillatoria limosa (AGARDH ex GOMONT) and two different morphotypes of Oscillatoria aff. tenuis. Analyses of geosmin in the mat confirmed that the algal mats dominated by Oscillatoria were geosmin-producers. An analysis of redundancy (RDA) based on algal and cyanobacterial taxa showed a gradient of velocity and dissolved geosmin in the water associated to the occurrence of the two Oscillatoria taxa, and allowed to summarise that low water velocity favours the development of these algal mats. Temperature also exerted a critical role for the geosmin occurrence in the river, since it contributed to determine the period of development of the algal mats in the different sites of the river.

The composition of algal species and pigments and the structural and functional characteristics of the algal community were investigated in an acid stream of southwestern Spain, the Rio Tinto. The algal community had low diversity and showed few seasonal differences. It was mainly made up of Klebsormidium flaccidum Kutz. (Silva, Mattox & Blackwell) that produced long greenish or purplish filaments, Pinnularia acoricola Hust. (producing brown patches) and Euglena mutabilis Schmitz. The algal filaments made up a consistent biofilm that also included fungal hyphae, iron bacterial sheaths, diatoms, and mineral particles. HPLC analyses on Rio Tinto samples showed that undegraded chl accounted for 67% of the total chl in the filamentous patches but were a minority in the brown patch (2.6%). The brown patch had a concentration of carotenoids eight times lower than that observed in the green patch. When chl concentrations were weighted for the proportion of the different patches on the streambed, undegraded chl a accounted for 89.2 mg

Fifteen European rivers and streams belonging to watersheds in Sweden, the Netherlands, and Spain respectively, were sampled by allowing periphyton to colonize submerged glass substrata. Their zinc tolerances were quantified in short-term laboratory tests, where inhibition of photosynthesis in microalgae and thymidine incorporation in bacterial DNA was measured, and expressed as EC50 values. The variability in zinc tolerances was high reaching 1.5-2.5 orders of magnitude, ranging from 25-8145 microM for photosynthesis and 15-467 microM for thymidine assays. Based on the observed variability, uncertainty factors were estimated for the extrapolation of zinc toxicity data from river to river, both regionally and interregionally. Under the assumption to protect 95% of the observed communities the regional uncertainty factors were 1.7-4.3 and the interregional 2.4-8.6. The sampling sites were characterized in terms of biotope physiography, water chemistry, periphyton biomass, trace element content, and species composition. Multivariate analysis of the data using PLS (Projection to Latent Structure), was used to generate hypotheses about the relation between periphyton zinc tolerance and the 123 so-called predictor variables. Zinc contamination, phosphate, nitrogen nutrients, pH, calcium, bicarbonate, dissolved organic carbon, and various diatom species are important predictors for zinc tolerance in the entire data set representing all 15 river stretches. Regional models suggested that very different factors determined the zinc tolerance in the Swedish and Dutch periphyton. The results are interpreted in terms of Pollution-Induced Community Tolerance (PICT) and the bioavailability of zinc.